This invention is in the field of spectrophotometric determinations of concentrations of analytes in samples. The invention further relates to methods of calibrating spectrophotometers. Particularly, the method relates to the calibration of spectrophotometric apparatus designed to measure interferents in serum and plasma.
Clinical laboratory tests are routinely performed on serum or plasma of whole blood. In a routine assay, red blood cells are separated from plasma by centrifugation. Red blood cells and various plasma proteins may also be separated from serum by clotting prior to centrifugation. Hemoglobin (Hb), light-scattering substances like lipid particles, bile pigments bilirubin (BR) and biliverdin (BV) are typical substances which will interfere with and affect spectrophotometric and other blood analytical measurements and are therefore referred to as interferents. The presence of such interferents affects the ability to perform tests on the serum or plasma and as such can be said to compromise specimen integrity.
Visual inspection can be used to determine the presence of interferents in serum and plasma but such a method relies on the experience and knowledge of the observer and is therefore unreliable. The use of an apparatus or instrument to measure interferents in serum and plasma i.e., assess specimen integrity, is a substitute for visual inspection and the interferents may be regarded as analytes with respect to the apparatus. Measurement of interferents are taught in WO 9838961 and WO 9839634. Because quantitative results from the determination of the concentration of such interferents are reported based on specific calibration algorithms, there is a need to calibrate and to monitor calibration performance daily.
Unlike many blood analytical apparatus, calibration of reagentless spectrophotometric apparatus used to measure the concentration of analytes or interferents in a serum or plasma sample is a cumbersome time intensive exercise. Each apparatus used for the purposes of determining the concentration of interferents must be calibrated according to procedures known in the art, for example, the process described herein, in the section titled xe2x80x9cPrimary Calibration,xe2x80x9d and over the lifetime of an apparatus can amount to a considerable amount of time and cost. Furthermore, in settings where a large number of apparatus is needed to perform multiple sample measurements (such as blood banks for example) the time required for calibration can become a real burden on the efficiency of the of the quality control process.
Martinek (J. Amer. Med. Technol., July-August 1978, p. 210-216) teaches a method of photometric correction, involving liquid absorbance standards to correct one spectrophotometer to match another using a slope and bias correction. This method may also be used for test methods that require reagents.
U.S. Pat. No. 4,866,644 teaches a method of calibrating a second apparatus to produce results for a test sample, as if the sample was tested on a first apparatus. The method combines photometric correction with a mathematical process that computes a waveshift for each index point. The waveshifts are derived from the assessment of readings determined for a plurality of samples on the two apparatus. The waveshifts are applied as corrections to an existing wavelength calibration table of the second apparatus in order to make the second apparatus behave in a manner similar to the first apparatus. In U.S. Pat. No. 4,866,644, the same wavelengths are assigned to the same corresponding index points in every instrument. Therefore, there is no derivation of a new wavelength calibration table of the second instrument, and the waveshift correction is applied to each measurement as it is determined on the second instrument
WO 94/08225 discloses a method involving the modification of the constants of a primary calibration algorithm of a second or recalibrated apparatus, to yield results consistent with a first apparatus that is in control. A limitation of this method is that the number of samples required must be at least one more than the number of terms used in the primary calibration equation, because a mathematical system of xe2x80x9csimultaneous equationsxe2x80x9d is used to generate a new constant for each term in the primary calibration algorithm. Furthermore, a predicted dependant variable, such as a chemical or physical property, of a calibrator is required to generate the new constants.
WO 97/47972 teaches a method for a second apparatus to produce results for a test sample, as if the sample was tested on the first apparatus involving testing a set of stable samples, whose absorbance spectra mimic that of the analytes, on both the first and a second apparatus, and predicting the analyte concentrations after applying a primary calibration algorithm. This method requires a predicted dependant variable, for example a chemical or physical property, of the calibrator. Analyte results predicted by both apparatus are used to perform a slope and bias correction of each analyte prediction on a test sample. The calibration set requires the property of having an absorbance spectra similar to the analyte.
There is a need for a method to simply and accurately calibrate a second apparatus, and to recalibrate a first or second apparatus that is no longer in control.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.
This invention is in the field of spectrophotometric determination of concentrations of analytes in samples. The invention further relates to methods of calibrating spectrophotometers. The method may be used for the calibration of spectrophotometric apparatus designed to measure interferents in serum and plasma. The invention also relates to a method of transferring calibration algorithms from a first apparatus to a second apparatus.
The present inventor has found that for a given analyte, a xe2x80x9cPrimary Calibration Algorithmxe2x80x9d developed for a xe2x80x9cFirst apparatusxe2x80x9d can be transferred onto a xe2x80x9cSecond Apparatusxe2x80x9d. Therefore, the Second Apparatus need not be subjected to the cumbersome, time intensive Primary Calibration process.
In one aspect of the invention, the First Apparatus that is known to be xe2x80x9cIn Controlxe2x80x9d is used to assign absorbance values to a xe2x80x9cSet of Calibratorsxe2x80x9d from a batch or lot, and any Second Apparatus can be calibrated rapidly by a process of xe2x80x9cCalibration Algorithm Transfer,xe2x80x9d and the concentration of an analyte in a sample determined by applying the xe2x80x9cPrimary Calibration Algorithmxe2x80x9d to a corrected interpolated absorbance measurement of the sample. Therefore, the present invention provides a method for calibrating a Second Apparatus using a Set of Calibrators with absorbances assigned by the First Apparatus.
In yet a further aspect of the invention a method for adjusting the absorbance of sample obtained on a second apparatus to normalize it with that of a first apparatus that is in control (xe2x80x9cphotometric correctionxe2x80x9d) using a xe2x80x9cLinear Regression Equationxe2x80x9d is also provided.
The present invention provides a method (A) for a Calibration Algorithm Transfer comprising:
(i) obtaining a first set of absorbance measurements of a set of calibrators on a First Apparatus that is in control at wavelengths from a first wavelength calibration table;
(ii) establishing a second wavelength calibration table on a second apparatus, the first and the second wavelength calibration table may be the same or different, and obtaining a second set of absorbance measurements of the set of calibrators on the Second Apparatus, at wavelengths from the second wavelength calibration table;
(iii) determining a first interpolated absorbance for the first absorbance measurements for at least one wavelength of a Standard Set of Wavelengths, and determining a second interpolated absorbance for the second absorbance measurements for the at least one wavelength of the Standard Set of Wavelengths,
(iv) deriving a First Linear Regression Equation for each of the at least one wavelength of the Standard Set of Wavelengths using the first and the second interpolated absorbance measurement;
(v) incorporating the First Linear Regression Equation and at least one Primary Calibration Algorithm onto the Second Apparatus.
In a further aspect of the invention there is provided a method (B) of determining the concentration of an analyte in a sample in a second apparatus comprising:
(a) performing a Calibration Algorithm Transfer, as defined above;
(b) measuring an absorbance of the sample on the second apparatus, and determining a sample interpolated absorbance for at least one wavelength of the Standard Set of wavelengths;
(c) adjusting the interpolated absorbance with the First Linear Regression Equation to obtain an Adjusted Interpolated Absorbance; and
(d) calculating a concentration for the analyte by applying at least one Primary Calibration Algorithm for the analyte to the Adjusted Interpolated Absorbance.
In yet a further aspect there is provided a method (C) to derive a standard set of wavelength from a wavelength calibration table wherein the wavelength calibration table for the first or second apparatus is obtained by:
(i) projecting a first electromagnetic radiation of known wavelength, onto a first pixel of a first linear diode array of the first apparatus or a second linear diode array of the second apparatus;
(ii) using a second electromagnetic radiation of known wavelength, the second electromagnetic radiation having a different wavelength than the first electromagnetic radiation, projecting the second electromagnetic radiation onto a second pixel of the first or second linear diode array;
(iii) identifying the first and second pixels within the first or second linear diode array;
(iv) calculating a pixeldispersion for the first and second linear diode array; and
(v) assigning a wavelength to each pixel within the first and second linear diode array to produce the wavelength calibration table using the pixeldispersion and either the first electromagnetic radiation of known wavelength, and the first pixel, or the second electromagnetic radiation of known wavelength and the second pixel.
The wavelength calibration table for the first apparatus may also be obtained by:
(a) projecting a known wavelength of electromagnetic radiation, onto a pixel in a linear diode array of the first apparatus;
(b) identifying the pixel number of the pixel;
(c) assigning a wavelength to each pixel within the linear diode array to produce the wavelength calibration table using a predetermined pixeldispersion, the known wavelength of electromagnetic radiation, and the pixel number.
The wavelength calibration table of a second apparatus may be similarly obtained by projecting the electromagnetic radiation of known wavelength onto a pixel of the linear diode array of the second apparatus having the same pixel number as that of the first apparatus. Alternatively, the electromagnetic radiation may be projected on a pixel having a different pixel number.
In another aspect of the invention, the standard set of wavelengths can be obtained by:
(A) establishing a set of wavelengths common to the wavelength calibration table of both the first and second apparatus; and
(B) selecting a range of wavelengths of the set of wavelengths, the range of wavelengths having wavelengths belonging to the standard set of wavelengths.
In yet a further aspect of the invention photometric correction is provided by the following equation:
AIA=(interpolated absorbancexe2x88x92y-intercept)/slope;
Wherein, xe2x80x9cAIAxe2x80x9d is the Adjusted Interpolated Absorbance, xe2x80x9cinterpolated absorbancexe2x80x9d is as determined in the step of measuring, see step (b), Method (B) as described above, and xe2x80x9cy-interceptxe2x80x9d and xe2x80x9cslopexe2x80x9d are obtained from the first linear regression equation, where the First Linear Regression Equation is derived from a plot of interpolated absorbance measurements, the first interpolated absorbance measurements on an X-axis, and the second interpolated absorbance measurements on a Y-axis, the First linear regression equation having a y-intercept and a slope.
In a further aspect of the invention, a Second Apparatus that was calibrated by xe2x80x9cCalibration Algorithm Transferxe2x80x9d but is no longer in control, can be xe2x80x9cRecalibratedxe2x80x9d using a Set of Calibrators that was assigned absorbances or absorbance values by the First Apparatus, which was known to be in control. The present invention also provides a method for Recalibration of the First Apparatus in the same way as any Second Apparatus.
In this aspect of the invention, the Calibrators are measured in the apparatus which is being Recalibrated and absorbances recorded using the same Standard Set of Wavelengths as used in the First Apparatus. New, or xe2x80x9cSecond Linear Regression Equationsxe2x80x9d are then developed for each relevant wavelength with the absorbance measurements from this Set of Calibrators versus the absorbance measurements assigned by the First Apparatus to the lot or batch of Calibrators. Each generated Second Linear Regression Equation having an intercept and slope, is then stored in the apparatus being Recalibrated. Interpolated absorbance measurements of actual samples in the Recalibrated apparatus are then adjusted using the Second Linear Regression Equation(s). The term xe2x80x9cSecond Linear Regressionxe2x80x9d applies to Recalibration (and also calibration as described below) of an apparatus and the term xe2x80x9cFirst Linear Regressionxe2x80x9d applies to Calibration Algorithm Transfer from a First Apparatus to a Second Apparatus.
Thus the invention also provides a method (D) for Recalibrating an apparatus that is no longer in control comprising:
(i) obtaining absorbance measurements of a set of calibrators on the apparatus, the set of calibrators having assigned absorbance values, the apparatus comprising a Primary Calibration Algorithm;
(ii) determining interpolated absorbance values for the absorbance measurements for at least one wavelength of a Standard Set of Wavelengths;
(iii) establishing a Second Linear Regression Equation in the apparatus, using the interpolated absorbance values and the assigned absorbance values; and
(iv) incorporating the Second Linear Regression Equation on the apparatus to produce a recalibrated apparatus.
The concentration of an analyte in a sample may also be obtained (Method E) in a Recalibrated apparatus by;
(a) recalibrating the apparatus
(b) measuring an absorbance measurement of the sample;
(c) deriving an interpolated absorbance for the absorbance measurement for at least one wavelength of the Standard Set of Wavelengths in the recalibrated apparatus;
(d) adjusting the interpolated absorbance measurement with the Second Linear Regression Equation to obtain an Adjusted Interpolated Absorbance; and
(e) calculating a concentration for the analyte by applying the Primary Calibration Algorithm for the analyte to the Adjusted Interpolated Absorbance.
Photometric correction may also be performed on recalibrated apparatuses using the following equation:
AIA=(interpolated absorbancexe2x88x92y-intercept)/slope;
wherein, xe2x80x9cAIAxe2x80x9d is Adjusted Interpolated Absorbance, xe2x80x9cinterpolated absorbancexe2x80x9d is as determined in the step of deriving (step (c)), Method (E) as described above, and xe2x80x9cy-interceptxe2x80x9d and xe2x80x9cslopexe2x80x9d are obtained from the Second Linear Regression Equation, where the Second Linear Regression Equation is derived from a plot of electronically stored assigned absorbance measurements on an X-axis, and the interpolated absorbance measurements obtained on the recalibrated apparatus on a Y-axis, the Second linear regression equation having a y-intercept and a slope.
In a further aspect of the invention there is provided a method (F) for calibrating an apparatus lacking a primary calibration algorithm the comprising:
(i) obtaining absorbance measurements of a Set of Calibrators on the apparatus, the apparatus lacking a primary calibration algorithm, and the set of calibrators having assigned absorbance values,
(ii) determining interpolated absorbance values for the absorbance measurements for at least one wavelength of a Standard Set of Wavelengths;
(iii) establishing a Second Linear Regression Equation in the apparatus, using the interpolated absorbance measurements and the assigned absorbance values; and
(iv) incorporating the Second Linear Regression Equation, and at least one Primary Calibration Algorithm on the apparatus, to produce a calibrated apparatus.
A calibrated apparatus may be used to determine (Method G) the concentration of an analyte by:
(a) calibrating the apparatus according to the method described above;
(b) measuring an absorbance value of the sample;
(c) deriving an interpolated absorbance from the absorbance value for at least one wavelength of the Standard Set of Wavelengths in the calibrated apparatus;
(d) adjusting the interpolated absorbance measurement with the Second Linear Regression Equation to obtain an Adjusted Interpolated Absorbance; and
(e) calculating a concentration for the analyte by applying the Primary Calibration Algorithm for the analyte to the Adjusted Interpolated Absorbance, and wherein in the step of adjusting the interpolated absorbance is obtained using the following equation:
AIA=(interpolated absorbancexe2x88x92y-intercept)/slope;
wherein, xe2x80x9cAIAxe2x80x9d is Adjusted Interpolated Absorbance, xe2x80x9cinterpolated absorbancexe2x80x9d is as determined in the step of deriving (step (c)) of method (G) as described above and xe2x80x9cy-interceptxe2x80x9d and xe2x80x9cslopexe2x80x9d are obtained from the Second Linear Regression Equation, where the Second Linear Regression Equation is derived from a plot of electronically stored assigned absorbance measurements on an X-axis, and the interpolated absorbance measurements obtained on the calibrated apparatus on a Y-axis, the Second linear regression equation having a y-intercept and a slope.
The inventor has also found that the process of Calibration Algorithm Transfer and subsequent determination of analyte concentration can be accomplished by using an order of derivative of the absorbance in the Primary Calibration Algorithm, where absorbance correction or xe2x80x9cPhotometric Correctionxe2x80x9d may not be necessary, provided that the order of derivative of absorbance used in the Primary Calibration Algorithm at the selected wavelength(s) does not contain significant inter-apparatus variability as may be seen in the absorbances at the same wavelength(s). Absorbance variability between apparatus can be minimized in certain xe2x80x9cSections of the Absorbance Spectra,xe2x80x9d by using an order of derivative of the absorbance.
Thus in another aspect of the invention there is provided a method (H) of determining the concentration of an Analyte in a Sample in a second apparatus comprising:
(i) incorporating at least one primary calibration algorithm that uses an order of derivative of absorbance obtained for at least one of a standard set of wavelengths, on the second apparatus;
(ii) measuring absorbance values of the sample at three or more wavelengths from a wavelength calibration table on the second apparatus;
(iii) determining interpolated absorbance values from the absorbance values for wavelengths from a standard set of wavelengths;
(iv) obtaining a derivative of the interpolated absorbance values, using the order of derivative; and
(v) calculating a concentration of the Analyte in the sample, by applying the Primary Calibration Algorithm to the derivative.
In another aspect of the invention there is provided a method wherein absorbance values may be assigned to a second batch of calibrators using an apparatus that is in control. In particular a xe2x80x9cSecond apparatusxe2x80x9d may be used wherein adjusted interpolated absorbances are assigned to the calibrators of the second batch.
According to another aspect of the invention the Primary Calibration Algorithms and the absorbance measurements of the calibrators made on the First Apparatus can be electronically stored. Thus in a further aspect of the invention there is provided a medium for storing instructions adapted to be executed by a processor to determine analyte concentration within a sample, the instructions comprising
i) at least one primary calibration algorithm;
ii) the assigned absorbances of a set of calibrators obtained from a first apparatus; and
iii) the identity of first apparatus used to obtain the at least one primary calibration algorithm and the assigned absorbances.
In yet a further aspect of the invention there is provided a kit comprising the set of calibrators and the medium with the stored instructions as described above.
In another aspect of the invention there is also provided an apparatus for determining analyte concentration of a sample comprising a spectrophotometer, a light source, a power supply, a sample holder, a circuit board, a primary calibration algorithm, and said first linear regression equation.
In a further aspect of the invention there is also provided a system for determining presence of an analyte comprising
i) means for transmitting electromagnetic radiation of one or more known wavelengths through a sample;
ii) means for detecting electromagnetic radiation after transmission through the sample;
iii) means for incorporating a primary calibration algorithm,
iv) means for storing a wavelength calibration table and a standard set of wavelengths;
v) means for deriving a first linear regression equation, a second linear regression equation, or both a first and a second linear regression equation;
vi) means for detecting presence or concentration of an analyte within the sample.
The present invention provides a method to provide a simple reliable method of using primary calibration algorithms in a second apparatus that do not require representative samples for which the apparatus was designed. Rather, the standard samples used to calibrate a second apparatus can be any stable samples that produce a range of absorbances at all relevant wavelengths.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.